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First published online 14 November 2007
doi: 10.1242/jcs.010686

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Dynamin 2 mediates fluid-phase micropinocytosis in epithelial cells

Mayo Clinic, Department of Biochemistry and Molecular Biology and the Miles and Shirley Fiterman Center for Digestive Diseases, Rochester, MN 55905, USA

View larger version (81K): [in this window] [in a new window]   Fig. 1. Microinjection of purified anti-dynamin antibodies reduces fluid-phase endocytosis. (A-H) Fluorescence micrographs of Clone 9 cells microinjected with either a control solution (injection buffer or anti-kinesin antibodies) or affinity-purified peptide antibodies targeting Dyn2 and subsequently assayed for the internalization of fluorescently conjugated transferrin (A,C,E,G) or dextran (B,D,F,H). Control cells microinjected with either injection buffer alone (A,B) or purified inhibitory anti-kinesin antibodies (C,D) showed no reduction in the internalization of either fluorescently conjugated transferrin (A,C) or dextran (B,D). By contrast, microinjection of cells with 7.2-8.6 mg/ml of purified polyclonal anti-dynamin antibodies either specific for Dyn2 (E,F) or against a conserved region found in all conventional dynamins, MC65 (G,H), attenuated receptor-mediated and fluid-phase endocytosis. (*) Indicates an injected cell. (I,J) Quantitation of transferrin (I) and dextran (J) internalization in Clone 9 cells microinjected with either buffer alone, anti-kinesin antibodies or anti-dynamin antibodies based on measurement of fluorescence intensity units. Values obtained from microinjected cells were normalized to those of surrounding uninjected cells. Results represent the average ± s.d. of 100 cells measured in each of two independent experiments. Bar, 10 µm.

View larger version (81K): [in this window] [in a new window]   Fig. 2. Fluid-phase endocytosis is reduced in cells depleted of Dyn2 protein using siRNA treatment. (A-C') Fluorescence micrographs of Clone 9 cells incubated with fluorescently conjugated dextran (A',B',C') following either mock (A,A') or Dyn2 siRNA (B-C') treatment. Dyn2 protein levels were analyzed by staining for Dyn2 using immunofluorescence (A,B,C). (A,A') No effect on fluid-phase endocytosis was observed when cells treated with transfection reagent alone for 72 hours were allowed to internalize fluorescently conjugated dextran for 60 minutes in low serum medium (0.2% FBS). (B,B') Cells transfected with a solution containing a pooled mixture of four distinct siRNA duplexes targeting Dyn2 and assayed 72 hours later, as for cells in A,A', showed a substantial reduction in Dyn2 staining (B) along with a marked reduction in the amount of dextran-positive cytoplasmic vesicles (B'). (C,C') Treatment of cells as for B,B' but in the presence of high serum medium (10% FBS): a more modest reduction in dextran internalization was observed than in low serum conditions. (*) Indicates a cell with reduced Dyn2 levels. (D) Quantitation of the amount of fluorescently conjugated dextran internalized by Clone 9 cells that had either been mock-treated for 72 hours, treated with a pooled mixture of Dyn2 siRNA duplexes for 72 or 96 hours, or treated with Dyn2 siRNA duplex #2 for either 72 or 96 hours, in the presence of low serum (black bars) or high serum (white bars) medium. Fluid-phase endocytosis was reduced in cells treated with either the pooled mixture of siRNA duplexes (Pool) or siRNA duplex #2 (#2), as compared with mock-treated cells, under both low serum (0.2% FBS) and high serum (10% FBS) conditions; however, the effect was more dramatic under lower serum conditions. Values obtained from siRNA-treated cells were normalized to those of mock-treated cells under similar serum conditions. Results represent the average ± s.d. of 50 cells measured in each of three independent experiments. Bar, 10 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 3. Disruption of Dyn2 function in epithelial cells stimulated with EGF does not prevent macropinocytic internalization of fluid. (A-B') Fluorescence micrographs of HeLa (A,A') or Clone 9 (B,B') cells treated with Dyn2 siRNA for 72-96 hours prior to incubation with fluorescently conjugated dextran for 30 minutes in low serum medium (A,B) or low serum medium plus 30 ng/ml EGF (A',B'). Cells with reduced Dyn2 levels (*) showed a marked reduction in fluid-phase endocytosis under unstimulated conditions, as compared with surrounding untransfected cells (A,B). Treatment of cells with EGF, however, induced a significant internalization of fluorescently conjugated dextran via the macropinocytic pathway, regardless of Dyn2 expression levels (A',B'). (C-D') Fluorescence micrographs of HeLa (C,C') or Clone 9 (D,D') cells microinjected with anti-Dyn2 antibodies as described in Fig. 1 and allowed to internalize fluorescently conjugated dextran under low serum conditions for 60 minutes in the absence (C,D) or presence (C',D') of EGF. As seen for Dyn2-siRNA-treated cells, dextran uptake was noticeably reduced in antibody-injected cells under low serum conditions (C,D), as compared with controls, whereas this effect was more modest in cells treated with EGF (C',D'). (E,F) Quantitation of internalized fluorescently conjugated dextran in Clone 9 (black bars) and HeLa (white bars) cells treated with Dyn2 siRNA (E) or microinjected with anti-Dyn2 antibodies (F) based on fluorescence intensity measurements. Values obtained from siRNA-treated cells were normalized to those of mock-treated cells under similar conditions, and values obtained from microinjected cells were normalized to those of surrounding uninjected cells. Results represent the average ± s.d. of 50 cells measured in each of three independent experiments. Bar, 10 µm.

View larger version (121K): [in this window] [in a new window]   Fig. 4. Inhibition or depletion of Dyn2 reduces internalization of the fluid-phase marker HRP. (A-B') Electron micrographs of HRP uptake in mouse hepatocytes (BNL CL.2) microinjected with control heat-inactivated antibody (A; A', enlargement of boxed area) or the anti-pan-dynamin antibody MC65 (B; B', enlargement of boxed area). Numerous HRP-positive endosomes were detected in control cells following a 60-minute incubation with HRP (A', arrows); however, cells microinjected with native antibody (MC65) displayed very few HRP-positive endocytic structures (B,B'), indicating inhibition of fluid-phase uptake. (C) Quantitation of HRP uptake in mock-treated (black bars) or Dyn2-siRNA-treated (white bars) Clone 9 cells under the indicated serum conditions based on HRP enzyme activity using o-phenylenediamine as a substrate. The ratio of HRP enzyme activity (absorbance at 490 nm) to protein concentration (absorbance at 600 nm) was determined for each sample and is presented as arbitrary units. The amount of internalized HRP was reduced in cells treated with Dyn2 siRNA under all serum conditions when compared with mock-treated cells under similar conditions, although stimulation with 10% FBS or with EGF appeared to induce Dyn2-independent mechanisms of fluid uptake. Results represent the average ± s.d. for three independent experiments.

View larger version (88K): [in this window] [in a new window]   Fig. 5. Differential distribution of wild-type and GTPase mutant Dyn2 spliced variants. (Top) Cartoon of the Dyn2 protein showing the tripartite GTP-binding motif (yellow) and the substitution/deletion sequences of the distinct spliced variants. Red lettering indicates amino acids that differ between the `a' and `b' Dyn2 spliced variants in the first splicing cassette. Other domains of the Dyn2 protein indicated are the pleckstrin homology domain (PH), guanine nucleotide exchange domain (GED) and proline-rich domain (PRD). (A,B,C) Fluorescence micrographs of Clone 9 cells expressing GFP-tagged wild-type (WT) versions of the indicated Dyn2 spliced variants (green) that were fixed and immunostained for clathrin (red). The different Dyn2 spliced variants show varying degrees of association with clathrin, both at the plasma membrane (insets) and at the perinuclear Golgi region (arrows). (A',B',C') Fluorescence micrographs of Clone 9 cells expressing untagged GTPase mutant (K44A) versions of the different Dyn2 spliced variants that were fixed and immunostained with anti-pan-dynamin (MC63, red) and anti-clathrin (green) antibodies. Two distinct phenotypes were generally observed, including either a perinuclear concentration of mutant Dyn2 protein (A',C', arrows) or long, linear, tubular structures terminating in a prominent clathrincoated tip (B'; arrowheads in inset). (*) Indicates a transfected cell. Bars, 10 µm.

View larger version (96K): [in this window] [in a new window]   Fig. 6. Inhibition of fluid-phase endocytosis in Clone 9 cells by specific Dyn2 spliced variant mutants. (A-C',E-G') Fluorescence micrographs of Clone 9 cells expressing either GFP-tagged wild-type (A, WT) or K44A GTPase mutant (B,C,E,F,G) forms of the different Dyn2 spliced variants that had been incubated with either fluorescently conjugated transferrin (A',B',C') or dextran (E',F',G'). Internalization of dextran was assayed under low serum conditions. Expression of GFP-tagged wild-type Dyn2(aa) (A) did not alter transferrin internalization (A'), whereas expression of the K44A mutant of this Dyn2 spliced variant (B) or the Dyn2(ab) spliced variant (C) did inhibit transferrin uptake (B',C'). By contrast, expression of Dyn2(aa)K44A (E) did not inhibit dextran uptake, even under low serum conditions (E'). However, expression of K44A mutant versions of two Dyn2 spliced variants, Dyn2(ba) (F) and Dyn2(bb) (G), did attenuate dextran internalization under low serum conditions (F',G'). (*) Indicates a transfected cell. (D,H) Quantitation of transferrin (D) and dextran (H) internalization in cells expressing wild-type (black bars) or K44A mutant (white bars) versions of the different Dyn2 spliced variants based on fluorescence intensity measurements. Although mutant forms of all of the Dyn2 spliced variants reduced the internalization of transferrin (D), the effects on dextran uptake (fluid-phase endocytosis) appeared to depend on the Dyn2 spliced variant being expressed (H). As occurred when other methods were used to inhibit Dyn2 function, the inhibitory effects of Dyn2(ba)K44A expression on the internalization of dextran were reduced when cells were assayed in the presence of 10% FBS (H, gray bars). Values obtained from cells expressing Dyn2 constructs were normalized to those of surrounding untransfected cells. Results represent the average ± s.d. of 50 cells measured in each of three independent experiments. Bars, 10 µm.

View larger version (91K): [in this window] [in a new window]   Fig. 7. Expression of either of two specific Dyn2 spliced variants, Dyn2(ba) or Dyn2(bb), rescues fluid-phase endocytosis in Dyn2-siRNA-treated cells. (A,A') Fluorescence micrographs of Clone 9 cells treated with transfection reagent alone (Mock) that were allowed to internalize fluorescently conjugated dextran for 60 minutes under low serum conditions and were subsequently immunostained for Dyn2. Mock-treated cells show a punctate plasma membrane and Golgi localization of Dyn2 (A), and a perinuclear accumulation of dextran-positive cytoplasmic vesicles (A'). (B-C') Clone 9 cells were treated with Dyn2 siRNA for 48 hours and then transfected with constructs encoding untagged versions of the different Dyn2 spliced variants. Twenty-four hours later, cells were assayed for dextran internalization and immunostained for Dyn2. Fluorescence micrographs of Dyn2-siRNA-treated cells re-expressing either Dyn2(ba) (B, asterisk) or Dyn2(bb) (C, asterisk) show that dextran uptake has been `rescued' in these cells (B',C'), whereas surrounding cells still depleted of Dyn2 internalize relatively little dextran. (D) Western blot analysis of Dyn2 protein levels in siRNA- and mock-treated cells performed in parallel with the rescue experiments. (E) Quantitation of dextran uptake in cells treated with reagent alone (Mock), Dyn2 siRNA, or Dyn2 siRNA followed by re-expression of the indicated Dyn2 spliced variants based on fluorescence intensity units. All images were acquired and adjusted equally, allowing for a comparison among all conditions. Results represent the average ± s.d. of 50 cells measured in each of two independent experiments.